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How about radiation-damaged/smashed Sulphur? You could test this by refining occupancy of the cys S. JPK +++++++++++++++++++++++++++++++++++++++++++++++++ Jacob Pearson Keller Research Scientist / Looger Lab HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 Desk: (571)209-4000 x3159 Cell: (301)592-7004 +++++++++++++++++++++++++++++++++++++++++++++++++ The content of this email is confidential and intended for the recipient specified in message only. It is strictly forbidden to share any part of this message with any third party, without a written consent of the sender. If you received this message by mistake, please reply to this message and follow with its deletion, so that we can ensure such a mistake does not occur in the future. From: CCP4 bulletin board <[log in to unmask]> On Behalf Of Lumbini Yadav Sent: Wednesday, July 10, 2019 7:12 AM To: [log in to unmask] Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue Thank you for the valuable input On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson <[log in to unmask]<mailto:[log in to unmask]>> wrote: Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S. And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or... But that still doesnt explain WHAT it is . Sorry not to be more help.. Eleanor On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav <[log in to unmask]<mailto:[log in to unmask]>> wrote: No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below . I do not seen density near cysteine that was visible in Fo-Fc map On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson <[log in to unmask]<mailto:[log in to unmask]>> wrote: The key word for refmac is ANOM MAPONLY Are you using GUI2? Eleanor On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav <[log in to unmask]<mailto:[log in to unmask]>> wrote: I have soaked my crystals in sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel <[log in to unmask]<mailto:[log in to unmask]>> wrote: Have you mass-speced the protein before crystallization to make sure it wasn’t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine (3-letter code: SCY). Best, Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin board [mailto:[log in to unmask]<mailto:[log in to unmask]>] On Behalf Of Lumbini Yadav Sent: Tuesday, July 09, 2019 5:22 AM To: [log in to unmask]<mailto:[log in to unmask]> Subject: [ccp4bb] Fo-Fc density close to cysteine residue Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma. Does anyone have an idea about what this density could be? Covalent modification? Thanks. 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